Conformation-specific anti-von Willebrand factor antibodies

ABSTRACT

The present invention is directed to antibodies and antibody fragments that bind specifically to the active conformation of human von Willebrand factor. Most preferred are recombinantly produced single chain variable immunoglobulin fragments. Because the antibodies or antibody fragments act only at the sites of thrombus formation and do not interfere with the normal activity of circulating platelets, they are particularly well suited for use as antithrombotic agents in a wide variety of applications.

FIELD OF THE INVENTION

[0001] The present invention is directed to compositions containing oneor more agents that recognize the active conformation of human vonWillebrand and that inhibit the interaction between this factor andplatelets. The compositions can be used either therapeutically orprophylactically to prevent thrombus formation in patients. Compositionsmay also be used diagnostically to detect sites where thrombosis islikely to occur.

BACKGROUND OF THE INVENTION

[0002] Thrombosis occurring at atherosclerotic plaques is a major causeof morbidity and mortality in the United States. The initial event inthrombus formation is the adhesion of platelets to an injured ordiseased arterial wall. Adhesion is enhanced and stabilized by a plasmaprotein, the von Willebrand Factor, which forms a bridge betweencomponents of the vessel wall and receptors on the platelet surface,primarily the glycoprotein Ib/IX/V complex. There are two uniquefeatures of this interaction that set it apart from adhesive eventsinvolving other cells and plasma proteins. First, the interaction of vWFwith platelets is the only reaction that permits platelets to remainattached to vessel walls under the high shear/high flow conditionspresent in arteries, arterioles, and capillaries. Second, vWF is theonly plasma adhesive protein which must undergo a conformation changebefore it is able to bind to its platelet receptor.

[0003] Attempts have been made to prevent thrombus formation byintroducing either antibodies (see, e.g., EP 747,060; WO 96/17078; andU.S. Pat. No. 5,336,618) or peptides (see, e.g., U.S. Pat. No.5,688,912; U.S. Pat. No. 5,493,007; U.S. Pat. No. 5,114,842; WO93/16712; and EP 319,315) that bind to platelet receptors. One problemwith the use of such agents is that they are nonselective—interferingwith the function of all circulating platelets. The development of anagent that acts specifically at sites of thrombosis to inhibit plateletadhesion would represent a clear advance in the treatment and preventionof stroke, myocardial infarction, and related conditions. Equallyimportant, such an agent might be used diagnostically to identify siteswhere blood vessels are at risk of becoming occluded.

SUMMARY OF THE INVENTION

[0004] Using recombinant DNA and phage display technology, murineanti-human vWF antibodies have been made which specifically recognizeactivated vWF and interfere with its ability to promote plateletadhesion. The antibodies act at sites of thrombus formation but do notbind to circulating, unactivated forms of vWF. This results inantithrombotic agents that are both safer and more efficacious.

[0005] In its first aspect, the invention is directed to a compositioncomprising an antibody that binds selectively to the active conformationof human vWF, thereby inhibiting its ability to interact with platelets.As used herein, “selective binding” means that an antibody has at leasta tenfold, and preferably at least a hundredfold, greater affinity forvWF when it is in its active conformation compared to when it isunactivated. Relative affinity can be determined using standard bindingassays in which vWF is examined both in the presence and absence of anactivating agent such as Ristocetin. Unless otherwise indicated, theterm “antibody” refers both to intact antibodies as well as tofragments, particularly to recombinantly engineered fragments, thatretain their ability to bind to antigen. Inhibition of platelet bindingoccurs whenever there is a statistically significant reduction in theamount of vWF-induced platelet aggregation in the presence of antibody.In the most preferred embodiment, compositions contain recombinantlyproduced single chain variable region (ScFv) fragments ofimmunoglobulins directed against a vWF-A1 epitope. Typically, the ScFvfragment will be derived from the mouse and compositions designed fortherapeutic administration will contain a pharmaceutically acceptablecarrier.

[0006] In a second aspect, the invention is directed to a method ofidentifying an ScFv fragment that binds selectively to the activeconformation of human vWF. The method involves immunizing an animal,preferably a mouse, with an immunogen (either a peptide or a nucleicacid encoding a peptide) derived from the A1 region of human vWF. Afterimmunization, mRNA is isolated from the animal and used to produce anScFv cDNA library in a bacteriophage capable of displaying thefragments. The library is then screened to identify phage expressing afragment that binds selectively to the active conformation of vWF.Binding may be determined directly, in the presence and absence of anagent inducing vWF to assume an active conformation, or by examining theinhibition of vWF-induced platelet aggregation. Once an appropriatephage has been identified, the DNA encoding the ScFv fragment may berecovered and subcloned in an expression vector. Finally, recombinantScFv is produced in a host cell transformed with the vector andpurified. The ScFv fragments obtained in this manner are part of theinvention.

[0007] The present invention is also directed to a method for preventingthrombus formation in a patient by administering a pharmaceuticalcomposition containing an antibody of the type discussed above, i. e.,an antibody binding selectively to the active conformation of human vWF.The pharmaceutical composition should be administered at a dosagesufficient to prevent the binding of activated vWF to platelets and maybe administered either therapeutically or prophylactically.Therapeutically, the composition may be administered to a patient withan occluded blood vessel either alone or in conjunction withthrombolytic agents such as tissue plasminogen activator orstreptokinase. Prophylactically, the composition may be administered topatients at risk of thrombosis due to atherosclerosis or during medicalprocedures that carry a risk of vessel occlusion, e.g., angioplasty,stent placement, or graft insertion.

[0008] Antibodies may also be detectably labeled and used in conjunctionwith imaging techniques to determine sites within the vasculature wherethrombosis is likely to occur, e.g., where there has been plaque ruptureor blood vessel damage. Because ScFv fragments are missing regions ofantibodies that are often responsible for nonspecific binding, thesefragments are preferred for all in vivo diagnostic procedures.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention is directed to antibodies that specificallyrecognize the activated conformation of vWF and prevent it frominteracting with platelets. It encompasses methods for making ScFvconformation-specific fragments and methods for using such antibodiesdiagnostically, therapeutically and prophylactically.

[0010] A. Antibodies Selectively Binding to the Active Conformation ofvWF

[0011] Methods for making and detecting antibodies have been describedin numerous standard reference works such as: Harlow et al., Antibodies,A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1988); Klein,Immunology: The Science of Self-Nonself Discrimination (1982); Kennettet al., Monoclonal Antibodies and Hybridomas: A New Dimension inBiological Analyses (1980); and Campbell, “Monoclonal AntibodyTechnology,” in Laboratory Techniques in Biochemistry and MolecularBiology (1984). The process for producing conformation-specificantibodies may involve either injecting the intact vWF protein into anappropriate animal or, preferably, injecting short peptides made tocorrespond to regions of vWF that are believed to interact withplatelets, i.e., peptides from the A1 domain. As an alternative, nucleicacids encoding vWF or portions of vWF may be administered to animals(see, U.S. Pat. No. 5,589,466; U.S. Pat. No. 5,580,859; and U.S. Pat.No. 5,703,055). The preferred animal for immunization is the mouse.

[0012] The term “antibody” refers to monoclonal antibodies, polyclonalantibodies and to fragments of these antibodies that continue to bind toantigen. Polyclonal antibodies are derived from the sera of animalsimmunized with the antigen. Monoclonal antibodies can be prepared usinghybridoma technology (Kohler et al., Nature 256:495 (1975); Hammerlinget al., in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.pp. 563-681 (1981)). In general, this technology involves immunizing ananimal (usually a mouse) with antigen, extracting splenocytes from theimmunized animal and then fusing the splenocytes with myeloma cells,e.g., SP₂O cells. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium and then cloned by limitingdilution (Wands et al., Gastroenterology 80:225-232 (1981)). The cellsobtained through such selection are then assayed to identify cloneswhich secrete antibodies capable of binding specifically to the activeconformation of vWF. Antigen-binding fragments may be produced byproteolytically cleaving intact antibodies using enzymes such as papain(to produce Fab fragments) or pepsin (to produced F(ab′)₂ fragments).

[0013] Assays appropriate for measuring the binding of antibody to vWFare well known in the art. For example, radioimmunoassays orimmunometric assays, also known as “two-site” or “sandwich” assays, maybe used (see Chard, “An Introduction to Radioimmune Assay and RelatedTechniques,” in Laboratory Techniques in Biochemistry and MolecularBiology, North Holland Publishing Co., N.Y. (1978)). In one variety ofsuch assays, the antibody to be tested is immobilized on a solid supportand then incubated with a solution containing detectably labeled vWF inthe presence and absence of an activator such as Ristocetin. Nonspecificbinding may be determined by carrying out parallel incubations in thepresence of an excess quantity of unlabeled vWF and activator. Thisshould be subtracted from total binding, i.e., binding in the absence ofunlabeled vWF, to arrive at the specific binding for each sample tested.Other steps such as washing, stirring, shaking, filtering, and the likemay be included in the assays as necessary. Typically, wash steps areincluded after the separation of bound ligand from ligand remaining insolution and prior to the quantitation of binding, e.g., by countingradioactive isotope. As an alternative, assays examining the ability ofantibodies to inhibit the aggregation of platelets in the presence ofactivated vWF may be used.

[0014] It is highly desirable that antibodies identified as binding tothe active conformation of vWF be reexamined in a concentration rangesufficient to perform a Scatchard analysis on the results. This type ofanalysis is well known in the art and can be used for determining theaffinity of an antibody for an antigen (see, e.g., Ausubel et al.,Current Protocols in Molecular Biology, pp. 11.2.1-11.2.19 (1993);Laboratory Techniques in Biochemistry and Molecular Biology, Work etal., N.Y. (1978)). Computer programs may be used to help in the analysisof results (see, e.g., Munson, P., Methods Eenzymol. 92:543-577 (1983);McPherson, “Kinetic, EBDA Ligand, Lowry—A Collection of RadioligandBinding Analysis Programs,” Elsevier-Biosoft, U.K. (19985)).

[0015] B. ScFv Fragments

[0016] ScFv fragments are proteins consisting of the V_(L) and V_(H)antibody polypeptide chains synthesized as a single chain with thecarboxyl terminus of V_(L) linked by a peptide bridge to the aminoterminus of V_(H). Methods for recombinantly producing these peptides inE. coli are well known in the art (see Bird et al., Science 242:423-426(1988); Huston et al., Proc. Nat'l. Acad. Sci. USA 85:5879-5883 (1988);and de Kruif et al., J. Mol. Biol. 248:97-105 (1995)). Although anymethod for generating these fragments is compatible with the presentinvention, the preferred method consists of immunizing mice withpeptides derived from the A1 region of vWF. After immunization, splenicmRNA is harvested from the mice and used to produce a cDNA library in abacteriophage which displays the ScFv fragments. Phage are then screenedto determine those that interact specifically with the activated form ofvWF. ScFv segments are recovered from these phage, incorporated into anexpression vector, and cloned in E. coli. The recombinant ScFv fragmentsproduced by the bacteria may be purified and further tested for bindingaffinity to both activated and unactivated vWF.

[0017] Using this procedure, recombinant antibody fragments have beenobtained that have three important characteristics: 1) they only bind tovWF that has been activated by prior immobilization or by exposure to anactivating agent like Ristocetin; 2) they inhibit the binding of vWF toplatelets as measured using a Ristocetin-induced platelet agglutinationassay; and 3) they inhibit flow-dependent platelet adhesion toimmobilize vWF. The selectivity of these fragments makes them suitablefor use in pharmaceutical compositions designed for administration topatients as antithrombotic agents.

[0018] C. Therapeutic and Diagnostic Use of Antibodies

[0019] Pharmaceutical compositions containing antibodies specific forthe active conformation of vWF may be used to treat or prevent coronaryarterial ischemic syndromes, including unstable angina and acutemyocardial infarction, as well as to treat cerebrovascular andperipheral vascular ischemia. The compositions may also be used inconjunction with therapeutic interventions such as stent placement,balloon angioplasty, or graft insertion.

[0020] Any route of administration and dosage form is compatible withthe present invention and conformation-specific antibodies may beadministered as either the sole activate agent or in combination withother therapeutically active drugs such as thrombolytics. In general,parenteral delivery using the intravenous, intraarterial, intramuscular,intraperitoneal, intracutaneous, or subcutaneous routes is preferred.

[0021] Dosage forms may be prepared using methods that are standard inthe art (see, e.g., Remington's Pharmaceutical Sciences, 16th ed., A.Oslo ed., Easton, Pa. (1980)). Active agents may be used in combinationwith any of the vehicles and excipients commonly employed inpharmaceutical preparations, e.g., talc, gum arabic, lactose, starch,magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils,paraffin derivatives, glycols, etc. Solutions can be prepared usingwater or physiologically compatible buffers, or organic solvents such asethanol, 1,2-propylene glycol, polyglycols, dimethyl sulfoxide, fattyalcohols, triglycerides, partial esters of glycerine, and the like.Preferred parenteral compositions may be prepared using conventionaltechniques and include sterile isotonic saline, water, 1,3-butanediol,ethanol, 1,2-propylene glycol, polyglycols mixed with water, Ringer'ssolution, etc.

[0022] The dosage of active agent to be administered to a patient willbe determined using methods well known in the art and will depend upon awide variety of clinical factors. By way of example, a typicalpharmaceutical composition for injection may comprise 1 ml of sterilebuffered water and 10 mg of antibody. A typical composition forintravenous infusion may comprise 250 ml of sterile Ringer's solutionand 10 mg of protein. The compositions may be administered eitherprophylactically or therapeutically. In therapeutic applications,compositions are administered to a patient suffering from a disease orcondition in an amount sufficient to produce a positive therapeuticeffect. For example, in the case of angina, dosage should be adjusted tothe point where pain is alleviated. For occluded vessels, it is expectedthat antibodies will be used in conjunction with one or morethrombolytic agents and dosage should be sufficient to achieve, at leastpartial, reperfusion.

[0023] Prophylactically, pharmaceutical compositions containing theconformation-specific antibodies are administered to a patient in orderto prevent the onset of an unwanted disease or condition. Thus,compositions may be administered to a patient with atheroscleroticplaques to prevent thrombosis or to patients undergoing therapeuticprocedures such as angioplasty to reduce the chance of vessel occlusion.

[0024] Antibodies may also be used diagnostically to identify sites ofpotential thrombus formation. This may be accomplished by labelingantibodies with an agent that is detectable by imaging techniques suchas NMR, MRI, or CAT scans. ScFv fragments should be especially useful inthis regard in that the portions of antibodies that are primarilyresponsible for nonspecific in vivo binding are not present in thesemolecules.

[0025] All references cited herein are fully incorporated by reference.Having now fully described the invention, it will be understood by thoseof skill in the art that the invention may be performed within a wideand equivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or embodiment thereof.

What is claimed is:
 1. A composition comprising an antibody that bindsselectively to the active conformation of human von Willebrand Factor(vWF) and wherein said antibody inhibits the binding of vWF toplatelets.
 2. The composition of claim 1, wherein said antibody is asingle chain variable region immunoglobulin (ScFv) fragment.
 3. Thecomposition of claim 2, wherein said ScFv fragment is from the mouse. 4.The composition of any one of claims 1-3, further comprising apharmaceutically acceptable carrier.
 5. A method of identifying a singlechain variable region immunoglobulin (ScFv) fragment that bindsselectively to the active conformation of vWF, comprising: a) immunizingan animal with an immunogen derived from the A1 region of human vWF; b)isolating mRNA from the immunized animal of step a); c) producing a cDNAlibrary from the isolated mRNA of step b) in a bacteriophage capable ofdisplaying ScFv fragments encoded by the cloned cDNA; and d) screeningthe library of step c) to identify a bacteriophage displaying a ScFvfragment that binds selectively to the active conformation of vWF. 6.The method of making and ScFv fragment that binds selectively to theactive conformation of vWF, comprising: a) immunizing an animal with animmunogen derived from the A1 region of human vWF; b) isolating mRNAfrom the immunized animal of step a); c) producing a cDNA library fromthe isolated mRNA of step b) in a bacteriophage capable of displayingScFv fragments encoded by the cloned cDNA; and d) screening the libraryof step c) to identify a bacteriophage displaying a ScFv fragment thatbinds selectively to the active conformation of vWF. e) subcloning therecombinant DNA encoding the ScFv fragment identified in step d) in anexpression vector; f) transforming a host cell capable of expressingrecombinant ScFv with the expression vector of step e); and g) purifyingthe recombinant ScFv produced by the host cell of step f).
 7. The methodof either claim 5 or 6, wherein the animal of step a) is a mouse.
 8. Themethod of either claim 5 or 6, wherein the screening of step d)comprises determining whether said ScFv fragment binds to vWF in thepresence and absence of an agent that induces vWF to assume an activeconformation.
 9. The method of claim 8, wherein said agent isRistocetin.
 10. The method of either claim 5 or 6, wherein the screeningof step d) comprises determining whether said ScFv fragment inhibits thebinding of vWF to platelets.
 11. An ScFv fragment produced by the methodof either claim 5 or
 6. 12. A method of inhibiting the binding of humanvWF to platelets, comprising contacting said human vWF with an antibodythat binds selectively to the active conformation of vWF.
 13. The methodof claim 12, wherein said antibody is a ScFv fragment.
 14. The method ofclaim 13, wherein said ScFv is from the mouse.
 15. A method ofpreventing thrombus formation in a patient, comprising administering apharmaceutical composition comprising an antibody that binds selectivelyto the active conformation of human vWF and that thereby prevents thebinding of vWF to platelets, wherein said pharmaceutical composition isadministered at a dosage sufficient to inhibit said thrombus formation.16. The method of claim 15, wherein said antibody is a ScFv fragment.17. The method of claim 16, wherein said ScFv fragment is from themouse.